Purification and characterization of fatty acid-binding proteins from rat heart and liver

Biochimica et Bioph.vsica Acta 837 (1985) 57-66

Elsevier BBA 52041

Purification and characterization of fatty acid-binding proteins from rat heart and liver Jan F.C. Glatz *, Anke M. Janssen, Camiel C.F. Baerwaldt and Jacques H. Veerkamp ** Deportment of Biochemistry, University of Nijmegen, P.0. Box 9101, 6500 HB Ngmegen (The Netherlands)

(Received May 28th. 1985)

Key words: Fatty acid-binding protein; Binding specificity; (Rat heart. liver)

Fatty acid-binding proteins were purified from delipidated cytosols of rat heart and liver by gel filtration and anion-exchange chromatography at pH 8.0 and by repeated gel filtration, respectively. Homogeneity of both proteins was demonstrated by a single band on polyacrylamide gels; each had a molecular weight of about 14000. Liver fatty acid-binding protein is more basic (pI,8.1) than that of heart (pI,7.0) and contains more basic amino acids. Examination of fatty acid binding by the binding proteins from heart and liver revealed the presence of a single class of fatty acid-binding sites in both cases with an apparent dissociation constant for palmitate of about 1 FM. Liver fatty acid-binding protein shows similar binding characteristics for palmitate, oleate aud arachidonate. Palmitate bound to heart fatty acid-binding protein was a good substrate for oxidation by rat heart mit~n~a. The results show that the fatty acid-biting proteins from rat heart and liver are closely related, but that they are distinct proteins. Introduction Fatty acid-binding protein is an abundant cytosolic protein of low molecular weight that is capable of binding fatty acids and some other lipidic compounds and therefore is considered to play an important role in cellular lipid metabolism (for review, see Ref. 1). Its precise nature and physiological function, however, have generally remained hypothetical. Fatty acid-binding protein may facilitate the cytosolic transport of fatty acids or be involved in their storage and thus behave as an intracellular counterpart of serum albumin, or may protect specific enzymes against inhibition by long-chain acyl-CoA esters. Fatty acid-binding

* Present address: Department of Human Nutrition, Agricultural University, Wage~nge~, The Netherlands. ** To whom correspondence should be addressed. 0005-2760/85/$03.30

protein has been investigated in a number of tissues, although most attention has been paid to the protein from rat liver. All proteins studied so far were found to have a similar molecular weight (11000-15 000) but showed isoelectric points varying from 4.8 to 7.6 [l]. In addition, several groups reported that purified rat liver fatty acidbinding protein preparations comprise three proteins with an identical M,, but with different pl values 12-51. The acidic forms may, however, represent denatured protein or arise from the binding of various kinds of ligands [4,6]. Binding of Iongchain fatty acids to bovine liver fatty acid-binding protein [7] and human serum albumin [S] was reported to be accompanied by a low pl value. Therefore, delipidation df the proteins appears to be an important prerequisite for the proper characterization of fatty acid-binding protein. Immunochemical cross-reactivity of fatty acidbinding proteins from different tissues has been

0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

58

observed. An antiserum raised against purified rat liver fatty acid-binding protein was reactive with the cytosol of intestine. adipose tissue, heart, skeletal muscle, kidney and testis [2,9,10], and that against rat jejunal’fatty acid-binding protein was reactive with the M, 12000 fraction of liver. adipose tissue and heart cytosol [9]. However, the fatty acid-binding proteins from human liver and adipose tissue are immunochemically different from those in the corresponding rat tissues. Two distinct fatty acid-binding proteins have recently been identified in rat intestine [12]. The possibility that still other closely related but immunochemitally distinct fatty acid-binding proteins are also present in the various tissues should. however. not be excluded. Recently, we showed that the fatty acid-binding capacity and fatty acid-binding protein content of rat heart cytosol is of similar magnitude as that of rat liver [13]. Earlier studies had not revealed the high fatty acid-binding protein content in heart, probably as a consequence of the use of inadequate binding assays [14] and of immunochemical assays in which antibodies to rat liver fatty acidbinding protein were used [2,9,10]. Fatty acidbinding proteins from rat and pig heart have so far only been studied by Fournier and co-workers [15-171 and recently that from rat heart also by Said and Schulz [18]. In order to compare their physicochemical properties we purified and characterized fatty acid-binding protein from cytosol of rat heart and liver. The results indicate that fatty acid-binding proteins from rat heart and liver are distinct proteins. Materials and Methods Preparation of delipidated proteins of I05 000 x g supernatant. Male albino Wistar rats, weighing 180-220 g, were used. The animals were fed ad libitum and killed during the light phase of the diurnal cycle (cf. Ref. 13). The liver and heart were perfused in situ with ice-cold buffer, consisting of 10 mM potassium phosphate (pH 7.4) and 154 mM KCl, and subsequently entirely removed. A 40% (liver) or 25% (heart) (w/v) homogenate was prepared in ice-cold perfusion buffer and was used to prepare a 105000 X g supernatant (cytosol). Details of these procedures have been given previ-

ously [13]. For each purification we usually used the tissues obtained from five to ten rats. The cytosolic proteins were delipidated by chromatography on a column of Lipidex (1 x 15 cm) at 37°C. exactly as described elsewhere [ 13.191, and stored at -20°C until use. Purification of liver frttty acid-binding protein. All steps in the purification of fatty acid-binding protein were performed at 4°C. The delipidated liver cytosolic proteins were applied to a column of Sephadex G-75 (1.6 X 85 cm). equilibrated with 30 mM Tris-HCI (pH 8.0), or a column of Sephacryl S-200 (2.6 X 110 cm), equilibrated with 30 mM Tris-HCI (pH 8.0) buffer containing 10%’ (v/v) glycerol, 1 mM dithiothreitol and 0.2 mM EDTA. The amounts applied were 40&100 and 200-250 mg protein, respectively. The flow rates were 25 and 40 ml/h and the fraction volumes 3.7 and 5.0 ml, respectively. Fractions containing fatty acid-binding protein activity were combined, concentrated by ultrafiltration using a Diaflo PM-10 or YM-2 membrane (Amicon Corporation. Lexington, MA, U.S.A.), and applied to a Bio-Gel P-10 column (1.8 x 75 cm), equilibrated with 30 mM Tris-HCl (pH 8.0). 10% (v/v) glycerol, 1 mM dithiothreitol, 0.2 mM EDTA. The flow rate was 8.4 ml/h, and 1.8-m] fractions were collected. Fatty acid-binding activity was detected in fractions 28-32. Purification of heart fatty acid-binding protein. The delipidated heart cytosolic proteins were fractionated on Sephadex G-75 or Sephacryl S-200 exactly as described for liver. Fatty acid-binding protein-containing fractions were combined, concentrated (see above) and applied to a DEAESephacel column (1.6 x 12 cm), equilibrated with 30 mM Tris-HCl (pH 8.0). The flow rate was 35 ml/h and the fraction volume 3.8 ml. The column was washed with the same buffer until unbound proteins had been completely eluted (Fig. 1). Retained protein was then eluted with a linear gradient (2 x 70 ml) of O-O.3 M KC1 in 30 mM Tris-HCI (pH 8.0). Ana(b~ical procedures. Protein was located in chromatographic fractions by measuring the A,,,, and was quantified (i) by the method of Lowry et al. [20] after the precipitation of protein with trichloroacetic acid [21] and using bovine serum albumin as standard, or (ii) by the spectrophoto-

59

metric method of Whitaket and Granum [ZZ], or (iii) from amino acid analysis (pure fatty acid-binding protein preparations only). Assay of palmitate binding was performed as described below, using 50-200 ~1 samples and 1 pM [l-‘4C]palmitate. Paimitate binding by 105 000 X g supernatant proteins was always determined in a small sample of the supernatant after dealbuminization by affinity chromatography at 4°C on a column of the immunoglobulin G fraction of rabbit anti-rat albumin antiserum coupled to Sepharose CL-4B after activation with tresylchlo~de [23]. Assay of fatty acid binding. For assay of fatty acid binding, protein samples were incubated in l&ml polyethylene vials in 10 mM potassiumphosphate buffer (pH 7.4) with various concentrations of [l-‘4C]palmitate. The final volume was 0.45 ml. After incubation for 10 min at 37°C the vials were cooled in ice. Unbound fatty acids were removed from the solution by adding 0.05 ml of an ice-cold, continuously stirred Lipidex buffer suspension (1: 1, v/v) and incubation for 10 min at 0°C. Fatty acid binding was calculated from the amount of radioactivity present in the supernatant after centrifugation of the vials and is expressed as pmol/pg of protein. Further details of this procedure and properties of the assay are described elsewhere [13,19]. E~ectr~p~oresis, is~e~e~tr~~focusing and amino acid analysis. Fatty acid-binding protein-contain-

ing fractions at various stages of purification were subjected to polyacrylamide slab gel electrophoresis in sodium dodecyl sulphate (SDS) (14.6% (w/v) acrylamide, 0.4% (w/v) bisacrylamide, and 0.1% (w/v) sodium dodecyl sulphate at pH 8.8) according to the method of Laemmli [24]. The Pharmacia Gel Electrophoresis Apparatus GE-2/4 LS was used (Pharmacia Fine Chemicals, Uppsala, Sweden). Each slot was loaded with about 5-25 pg of protein. Gels were stained with 0.2% Coomassie brilliant blue R-250 (Merck, Darmstadt, F.R.G.) in methanol/acetic acid/water (50 : 7 : 43, by vol.} and destained with methanol/acetic acid/water (5 : 7 : 88, by vol.). Isoelectric focusing of the purified fatty acidbinding protein preparations was carried out in gel rods in the presence of 6 M ureum, either exactly as outlined by Van Kleef and Hoenders [25], using a Desaga Electrophoresis Apparatus (Desaga,

Heidelberg, F.R.G.), or by a different method, using the same apparatus as for gel electrophoresis and Pharmalyte ampholytes (pH 3-10; Pharmacia Fine Chemicals, Uppsala, Sweden). In the latter case, the anode and cathode solutions were 10 mM phosphoric acid and 20 mM NaOH, respectively. Protein samples (5-10 pg per gel rod) were subjected to focusing at 400 V. The gel rods were stained with 0.1% Coomassie brilliant blue R-250 in ethanol/acetic acid/water (25 : 8 : 67, by vol.) and destained with the same solution without the dye. For determination of the amino acid composition, samples of purified fatty acid-binding proteins were hydrolyzed in duplicate with 6 M HCl at 105’C for 24 h and analyzed on a Rank Hilger Chromaspek analyzer, using a lithium-citrate buffer system and norleucine as the internal standard. Fatty acid oxidation. The oxidation of 20 PM [l-r4C]palmitate, bound to albumin or to purified fatty acid-binding protein, by rat heart mitochondria was measured after 5 min incubation as described elsewhere [26], and calculated from the production of ‘“CO, and l4 C-labeled acid-soluble products. Materials. Lipidex 1000 was kindly provided by Packard Instrument Co., Downers Grove, IL, U.S.A.; l-‘4C-labeled fatty acids were purchased from Amersham International, U.K., The origin of other materials is stated elsewhere [13]. Results P~ri~cati~n of fatty acid-birding protein from rat heart and fiver

The first step in the purification of fatty acidbinding protein from delipidated cytosolic proteins of rat heart and liver was gel filtration on Sephadex G-75 or Sephacryl S-200. Fatty acidbinding activity was, with both preparations, present mainly in fractions corresponding to a molecular weight of 10000-15 000, but some activity was also found in the region of M, 1500-2000. Elution profiles have been given previously [ 13,271. The latter fractions, which contain a fatty acid-binding peptide [28-301, were not studied further. With rat heart cytosol, fatty acid-binding activity was also usually present in the high J4, region

60

The latter protein could be eluted from the column by applying a linear gradient of KCl. These observations already indicate that liver fatty acid-binding protein is a basic protein that has a higher pl value than that of heart. Selected column fractions t~o;n both purification steps were subjected to polyacrylamide gel electrophoresis in SDS at pH 8.8. Both the fractions obtained after gel filtration of heart and liver cytosolic proteins showed essentially two major protein bands, corresponding to M, 14000 and 17 000 for heart and to A4, 14000 and 26 000 for liver (Fig. 2, lanes 3 and 7). After further purification on DEAE-Sephacel, heart fatty acid-binding nrotein showed a single band on the gel, corre-

HEART

LIVER

4 -3

Froctm

(x10number

Fig. 1. Purification of fatty acid-binding protein from rat heart (above) and liver (below) and DEAE-Sephacel. The combined, fatty acid-binding protein-containing fractions of the Sephadex G-75 column were chromatographed on DEAE-Sephacel with 30 mM Tris-HCI, pH 8.0 (up to fraction 36) and subsequently with a linear gradient of O-O.3 M KCI in the same buffer. Fractions were assayed for protein by measuring A *so ( -) and for palmitate binding (0). In order to trace myoglobin, the protein fractions from heart were also measured at 419 nm. Absorbance at this wavelength was only found with fractions 18-23. The results of representative experiments are shown, in which 1.6 mg (heart) and 2.9 mg (liver) of protein were applied to the column.

(M, > SOOOO), due to the presence of residual albumin (cf. Ref. 13). This activity peak was, however, always completely separated from the fatty acid-binding protein-containing fractions (M, 10 000-20 000 proteins). With the Sephadex G-75 and Sephacryl S-200 columns we obtained similar results, but the latter column had a higher capacity. For further purification we tested the use of anion-exchange chromatography. The combined fatty acid-binding protein-containing fractions of the gel filtration step were applied to a column of DEAE-Sephacel at pH 8.0. Fatty acid-binding protein from rat liver was not retained by the column, in contrast to that from heart (Fig. 1).

93

68 5L L5

26

20 172

1

2

3

4

5

67

89

Fig. 2. Polyacrylamide slab gel electrophoresis in SDS at pH 8.8 of fatty acid-binding protein-containing fractions from rat heart (lanes 2-5) and liver (lanes 7-9) at various stages of purification. Lane 1. calibration proteins (with M,): phosphorylase a (93000). bovine serum albumin (68000). leucine aminopeptidase (54000). ovalbumin (45000) cy-chymotrypsinogen A (26000). A, chain of calf a-crystallin (20000). myoglobin (17000) and cytochrome c (12400); 2, delipidated heart cytosol (25 pg protein); 3, combined fatty acid-binding protein-containing fractions after Sephadex G-75 chromatography (25 pg protein); 4, fraction 20 from DEAE-Sephacel column (10 pg protein); 5, fraction 41 from DEAE-Sephacel column (7 pg protein); 6, calibration proteins (see 1); 7, combined fatty acid-binding protein-containing fractions after Sephacryl S-200 chromatography (5 pg protein); 8. fraction 21 from DEAE-Sephacel column (5 p”g protein); 9. fraction 30 from Bio-Gel P-10 column (5 pg protein).

61

sponding to M, 14000 (Fig. 2, lane 5). The latter value is in agreement with observations by others [15,18]. The protein fraction not retained by the DEAE-Sephacel column at pH 8.0 mainly comprises myoglobin, as judged by gel electrophoresis (Fig. 2, lane 4) and also by its specific absorption at 419 nm (Fig. 1). The chromatographic behaviour corresponds to the reported pl values (7.5-8.5) for myoglobin from other animal species [31,32]. In contrast to heart, gel electrophoresis of the purified liver fatty acid-binding protein obtained after DEAE-Sephacel chromatography still revealed the presence of the two protein bands, indicating that in this case anion-exchange chromatography is not a suitable purification step. Homogeneous fatty acid-binding protein could, however, be obtained, when Sephacryl S-200 chromatography was followed by fractionation on a Bio-Gel P-10 column (Fig. 2, lane 9, and Fig. 3). Similar results were also recently reported by Haunerland et al. [7] for bovine liver fatty acidbinding protein. A summary of the purification procedures for fatty acid-binding protein from rat heart and liver, as routinely carried out, is given in Table I. To calculate the specific palmitate binding of pooled fatty acid-binding protein-containing fractions, precise quantification of protein is obligatory. The

TABLE

0.4-

-3000 =

F 8

$ 5

1 ,’

2

03.

$ s

0.2-

I I ’ I I 1 I ’

2 8 2

-2000 -$ .F 2 -1000 4

OlO-

E0 tt F 0

10

20

30

LO

/ 50

OF FATTY

ACID-BINDING

PROTEIN

Results are representative for four (heart) and chromatography gave protein losses of lo-25%. Purification

step

three

Protein (mg)

z

Fig. 3. Gel filtration on Bio-Gel P-10 (1.8~75 cm) of the pooled fatty acid-binding protein-containing fractions (3.2 mg protein) obtained after Sephacryl S-200 chromatography of liver cytosol. Elution was performed at 4°C with 30 mM Tris-HCI (pH 8.0). 10% (v/v) glycerol, 1 mM dithiothreitol, 0.2 mM EDTA. Fractions were assayed for protein by measuring A 2x0 ( -) and for palmitate binding (0). For the latter, 20- to 50-pl samples and 1 PM [l-‘4C]palmitate were used. Arrows indicate the elution positions of calibration proteins: A. cy-chymotrypsinogen; B, horse skeletal muscle myoglobin. The results shown are representative of three experiments.

method of Lowry et al. [20] appeared inadequate for following purification, due to the changing protein composition. With purified fatty acidbinding protein this method gave protein concentrations that were about 1.2- (heart) and 2.4(liver) times higher than the actual fatty acid-binding protein aminoacyl mass calculated on the basis

FROM

RAT HEART

(liver) purifications.

AND

LIVER

The concentrating

procedure

Specific palmitate binding a

Recovery of binding activity

of the samples

Purification (-fold)

(PmoVpg)

@I

Heart

105 000 X g supernatant Sephadex G-75 DEAE-Sephacel

28.9 h 11.8 ’ 1.6 ’

2.0 d 4.3 18.4

100 87 48

1 2.1 9.2

Liver

105000 X g supernatant Sephacryl S-200 Bio-Gel P-10

244b 4.9 c 0.8 ’

2.3 d 11.5 50.9

100 10 8

1 5.1 22.4

a ’ ’ d

0

Fraction number

I

PURIFICATION

Tissue

-

Measured with 1 gM [l-‘4C]palmitate at 37OC. Determined by the method of Lowry et al. [20]. Determined by the method of Whitaker and Granum [22] or from amino acid analysis Determined with part of the supernatant after dealbuminization.

(pure preparation

only).

prior

to

62

Physicochemical charucterizution of jLltt_).acid-hinding from heart and liver The molecular weights of the purified fatty acid-binding protein preparations were estimated by polyacrylamide gel electrophoresis (Fig. 2) and found to be 13 500 for heart and about 14000 for liver fatty acid-binding protein. These values are in accordance with those reported by others (listed in Ref. 1). When purified heart and liver fatty acid-binding protein were subjected to isoelectric focusing, single bands were observed at approx. pH 7.0 and 8.1. respectively (results not shown). The latter observations are consistent with the behaviour of the proteins on DEAE-Sephacel chromatography. The amino acid composition of liver fatty acidbinding protein (Table III) is in good agreement with published data (listed in Ref. 1). The composition of heart fatty acid-binding protein has not yet been reported and appears different from that of the liver protein and intestinal fatty acid-binding protein [35]. The larger number of basic residues of liver fatty acid-binding protein in comparison to that of heart also indicates a higher isoelectric point for the former protein.

of amino acid analysis (Table II). This discrepancy reflects differences between fatty acid-binding protein and the albumin standard in the relative abundance of Lowry-reactive residues. For liver fatty acid-binding protein similar differences were found by Ockner et al. [33]. Fatty acid-binding protein concentrations determined with the spectrophotometric method of Whitaker and Granum [22] were found to be in close agreement with the actual fatty acid-binding protein mass (Table II), making this simple and non-invasive method highly feasible for following the purification procedure. For liver fatty acid-binding protein, a final purification factor of 22 was obtained (Table I). This factor is in accordance with the hepatic content of fatty acid-binding protein (during the light phase of the light cycle) of about 4-50/o of the cytosolic proteins [13,33]. For heart fatty acidbinding protein, the final purification factor is lower, although the cytosolic content of fatty acid-binding protein in this tissue appears comparable to that of liver [13]. Since the presence of contaminating proteins in the heart fatty acid-binding protein preparation could not be demonstrated by gel electrophoresis (Fig. 2) or isoelectric focusing (see below), some loss of functional activity must have occurred during the purification procedure. The recovery of binding activity during purification was 48% for heart fatty acid-binding protein, but only 8% for that of liver (Table I). Purification yields for fatty acid-binding proteins have so far only been presented by Senjo et al. [34], who found a yield of 0.9% for rat brain fatty acid-binding protein. TABLE

Fatty acid binding by fatty ucid-binding protein from heart und liver The amount of various fatty acids bound to fatty acid-binding protein from heart as well as from liver was proportional to the amount of protein up to the binding of 40%50% of the added amount of fatty acid (results not shown), similar to earlier observations with serum albumin [19] and

II

INFLUENCE OF THE ASSAY PROCEDURE ON THE PROTEIN PURIFIED FATTY ACID-BINDING PROTEIN

CONTENT

OF PREPARATIONS

OF CYTOSOL

AND OF

Values are given in percentages of the prqtein content measured by the Lowry method [20] (arbitrarily set at 100) and represent means + S.D. of the number of preparations indicated within parentheses. FABP. fatty acid-binding protein. 105000

Method

Lowry et al. [20] h Whitaker and Granum Amino acid analysis

(221

X g

Purified

supernatant

FABP preparation

’

heart

liver

heart

liver

100 80+5(16)

loo IOOk 12(3)

100 7616(3) 89: 78

100 46*3(3) 41 * 8(3)

a Purified to homogeneity by the procedures h Using bovine serum albumin as standard.

outlined

in Table I.

63 TABLE

III

AMINO ACID COMPOSITION OF FATTY ACID-BINDING PROTEINS FROM RAT HEART AND LIVER * Values represent means+ SD. for three different fatty acidbinding protein (FABP) preparations. nd. not determined. Amino acid

Asx Thr Ser Glx Pro GIY Ala CYS Val Met Be Leu TYr Phe His LYS Arg Trp

Residues/1000

amino acid residues

heart FABP

liver FABP

120*1 130*5 71+4 106+2 11+1 loOi

102f s3* 601t 138+ 23rtll \ 115&

58*3 0 83+2 8&l 38+1 73+3 12+2 42+1 30+4 91+5 25*5 n.d.

47+ 791 12* 54* 52rdr 20f 46k 22j, 125* 265 n.d.

3 2 4 3 6 3 3 1 I 1 2 1 1 7 2

Fig. 4. Scatchard analysis of the binding of palmitate by purified fatty acid-binding protein (FABP) from rat heart (left) and liver (right). Fixed amounts of freshly purified fatty acidbinding protein (5 gg of the heart and 1.8 pg of the liver protein) were incubated with 0.1-3 PM [l-‘4C]palmitate in a total volume of 0.5 ml buffer solution. After equilibration. protein-bound and unbound palmitate were separated by the use of Lipidex at O’C. The concentration of free fatty acids was calculated from the initial concentration and the amount of protein-bound fatty acid. Protein was quantified by amino acid analysis. The results shown are representative for three fatty acid-binding protein preparations from each tissue.

delipidated cytosolic proteins from both tissues [13]. In contrast to cytosolic protein preparations 1131, palmitate binding activity of purified fatty acid-binding protein slowly decreased upon storage of the protein samples at -20°C in buffer solution (10 mM potassium phosphate, pH 7.4) with or without 10% (v/v) glycerol, but especially after lyophilization. Loss of functional activity occurred more with heart fatty acid-binding protein (about 30% loss after 2 weeks) than with liver fatty acid-binding protein (about 10% loss after 1 month). When fatty acid binding to the binding proteins was studied as a function of the total fatty acid concentration and the binding isotherms were analyzed by means of a Scatchard plot [36], a single class of saturable binding sites was observed with the proteins from heart and from liver (Fig. 4). With liver fatty acid-binding protein the apparent dissociation constants ( Kd) for palmitate, oleate and arachidonate were all calculated to be about 1 PM (Table IV). The value for palmitate is identical to that earlier found to delipidated cytosolic proteins of rat liver (1.04 k 0.15 ,uM (n = 3); Ref. 13). The binding capacity of liver fatty acidbinding protein was rather variable for all three fatty acids (Table IV), partly caused by the low amounts of pure fatty acid-binding protein available and difficulties with protein quantification. However, the results indicate the presence of one fatty acid-binding site per protein molecule, as was previously suggested [19]. With freshly purified heart fatty acid-binding protein the apparent K, for palmitate (about 1 PM (Table IV)) appeared comparable to the value earlier observed for delipidated cytosolic proteins of rat heart (0.83 f 0.11 FM (n = 4); Ref. 13). Due to loss of binding activity during delipidation and purification, the binding capacity found for heart fatty acid-binding protein will have been underestimated. This may cause the low fatty acid-toprotein ratio (Table IV). Purified pig heart fatty acid-binding protein showed a maximal fatty acidto-protein ratio of 0.2, as measured by electron spin resonance spectroscopy [16]. We also studied the oxidation of [1-‘4C]palmitate bound to (freshly) purified heart fatty acidbinding protein or to bovine serum albumin by isolated rat heart ~t~hond~a. Binding of [l-

64 TABLE

IV

FATTY LIVER

ACID-BINDING

PARAMETERS

OF PURIFIED

FATTY

ACID-BINDING

Data are derived from Scatchard analysis of individual binding isotherms. as shown used per assay were l-5 pg (based on amino acid analysis). Values represent means* protein preparations measured in triplicate.

PROTEINS

FROhl

RA’I IIEART

AND

for palmitate in Fig. 4. The amounts of protein S.D. for three freshly purified fatty acid-binding

--._._ Tissue

Hear: Liver

Fatty acid

palmitate palmitate oleate arachidonate

B,,,,,

Apparent

pmol/pg

(PM)

protein

mol/mol O.61 * 0.04 1.1f5+0.41 1.34kO.33 1.07 + 0.36

45j 3 83F30 94223 77_t?5

,’

K,,

1.07 i 0. IO 1.04+o.i1

1.03i 0.03 0.92 _c0.1 1 --~

’ Based on a molecular

weight for heart

fatty acid-binding

protein

“C]palmitate to the proteins was indicated by coelution from a column of Lipidex at 0°C (cf. Ref. 19). Palmitate bound to fatty acid-binding protein (molar ratio, 0.6: 1) appeared to be a somewhat better substrate for mitochondrial ,Roxidation than albumin-bound palmitate (molar ratio, 1 : 1); the oxidation rates were 10.3 & 2.1 and 7.9 rf 0.9 nmol/min per mg protein. respectively (means A-SD. of three determinations). This difference may be the result of the slightly lower palmitate binding affinity of fatty acid-binding protein (K, = 1 PM; Table IV) when compared to albumin at this molar ratio (Kd = 0.2 PM; Ref. 38). or of differences between the two proteins in their interaction with the mitochondrial membrane. These experiments show the capacity of fatty acid-binding protein to release fatty acids to iI~tracellular membranes, as earlier observed with electron spin resonance spectroscopy studies 1151, and make its assumed carrier function plausible. Competition between fatty acid-binding protein and albumin was also observed, since increasing amounts of fatty acid-binding protein markedly decreased the oxidation of albumin-bound palmitate (data not shown). Discussion Purification of the fatty acid-binding proteins from rat heart and liver and the subsequent study of their structural and functional properties revealed that these two proteins are closely related but essentially different proteins. Fatty acid-bind-

of 14000 and for liver fatty acid-binding

protein

of 13952 1371.

ing proteins from heart and liver have a similar molecular weight and also show similar palmitatebinding properties. However, liver fatty acid-binding protein appears a more basic protein than that from heart, which is also reflected in differences in amino acid composition between the two proteins. These observations provide a likely explanation for the relatively low or absent reactivity of an antiserum raised against purified liver fatty acidbinding protein with the cytosol of heart [2,9,10], when compared to the similar magnitude of the fatty acid-binding capacities of the two tissues [13]. The non-identity of the two fatty acid-binding proteins was also recently reported by others [18]. The low immunochemical reaction may be caused by a low degree of cross-reactivity of heart fatty acid-binding protein with anti-liver fatty acid-binding protein antibodies. or even by the presence of a small amount of liver fatty acidbinding protein in heart, as in intestine 1121. Very recently Fournier and Rahim 1391 determined the fatty acid-binding protein concentration in heart homogenate of adult male rats. by means of rocket immunoelectrophoresis with an antibody against purified rat heart fatty acid-binding protein. and found a content of 4.3 mg per g tissue. This value is higher than our data determined with the functional assay on isolated cytosol (3.5 pmol/pg protein in the light phase or 0.98 mg/g heart: Ref. 13). This may be due to subcellular binding of fatty acid-binding protein to organelles, but also methodological differences may be responsible. The purification of fatty acid-binding protein

65

from rat heart and liver cytosol to apparent homogeneity was achieved by repeated gel filtration only (liver) or by gel filtration and anion-exchange chromatography (heart}. Prior delipidation of the cytosol and the use of a specific and accurate binding assay allowed a more sensitive and precise detection of functional activity of fatty acid-binding protein in column fractions than the classical method by which fatty acid-binding protein is detected by coelution of radioactively labeled fatty acids [14,40]. The physicochemical properties of liver fatty acid-binding protein purified by the present method closely agree with published findings, except for its isoelectric point, which is higher than the values reported by most other investigators [2,4,5,33,41]. This difference is most likely caused by the absence of endogeneous fatty acids in our samples, since defatted preparations of bovine liver fatty acid-binding protein [7] and of human serum albumin [8] showed a higher pl value than non-defatted samples. For heart fatty acid-binding protein, we also found a higher isoelectric point than reported by Fournier et al. [15] for the delipidated protein (pl 5.0). However, the delipidation procedure (treatment with 20% (v/v) butanol) used by these workers may be denaturing for fatty acid-binding protein [4]. One of the difficulties with the purification of heart fatty acid-binding protein is its separation from myoglobin, which is present in relatively large quantities in rat heart (about 0.2-0.4 pmol/g wet weight tissue; Refs. 42 and 43) and which also has a low molecular weight (M, 16 400). Because of the high pZ value of myoglobin, complete separation of the two proteins could be achieved on anion-exchange chromatography at pH 8.0, as was also observed by Fournier et al. 1151.Contamination of myoglobin with fatty acidbinding protein may explain the observed fatty acid binding to this protein after gel filtration [44]. The affinity for palmitate binding of the purified liver fatty acid-binding protein is of similar magnitude as that of delipidated liver cytosolic proteins. The presence in the cytosol of large amounts of contaminating proteins apparently does not influence the fatty acid binding by fatty acid-binding protein. Fatty acid-binding characteristics of those binding proteins in the liver were found to be similar for palmitate, oleate and

arachidonate, being consistent with the idea, based on coelution experiments, that the fatty acid binding increases with chain length but decreases with the degree of unsaturation of the fatty acid [ 14,28,29]. In recent years it has been estabiished that distinct forms of fatty acid-binding protein exist. Rat intestine contains two fatty acid-binding proteins, one of which is identical to liver fatty acidbinding protein. while the other is confined to intestinal epithelium [12]. Our present study shows that rat heart contains still another distinct type of fatty acid-binding protein. It remains to be established whether the fatty acid-binding proteins identified in other tissues, such as adipose tissue [ll] and brain [34] are also different proteins. The various fatty acid-binding proteins may perform different functions in the cellular metabolism of fatty acids, as is indicated by the different regulatory responses of the two fatty acid-binding proteins in rat intestine [12]. Recently, a low concentration of the mRNAs of both intestinal and liver fatty acid-binding protein was detected in rat heart [45J. Immunochemical and functional assay for fatty acid-binding protein will help to establish the relative composition of its various forms of and its possible modulation in a certain tissue. Acknowledgements

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